In vitro Activation Prior to Transplantation of Human Ovarian Tissue: Is It Truly Effective?

Abstract

Research Question: What are the true benefits, if any, of disrupting the Hippo signaling pathway and stimulating the Akt pathway in xenotransplanted human ovarian tissue using an in vitro activation (IVA) approach? Design: Human ovarian tissue was retrieved from 18 young patients by laparoscopy and grafted to 54 severe combined immunodeficient mice. The experiment was conducted using fresh ovarian tissue (group I; n = 6 women), slow-frozen-thawed ovarian tissue (group II; n = 6 women), and vitrified-warmed ovarian tissue (group III; n = 6 women). Slow-freezing and vitrification procedures were performed according to Gosden's and Kawamura's protocols, respectively. The tissue (fresh, slow-frozen, and vitrified) was fragmented into small cubes (1 × 1 × 1 mm) to disrupt the Hippo signaling pathway and cultured or not in IVA medium for 48 h with Akt stimulators (PI3K stimulator and PTEN inhibitor), before being transplanted to the mice. All the grafts were maintained for 28 days. Results: (1) Follicular density: Follicular density decreased in all groups after transplantation, most significantly in the vitrification group. Culture with IVA had no impact. (2) Follicle activation : Addition of PI3K stimulator and PTEN inhibitor for 48 h prior to grafting did not significantly change the proportion of primordial follicles in any of the groups (fresh, slow-frozen, or vitrified tissue) compared to 48 h of control culture without these molecules. Particularly, vitrification and culture in IVA medium yielded no benefits in terms of growing follicle percentages or follicle proliferation rates. The large proportion of growing follicles in the vitrified tissue group after grafting may have been responsible for the higher rate of atresia. Conclusion: We were unable to demonstrate any significant benefits of cutting ovarian tissue into small cubes and applying IVA with Akt stimulators. The association of vitrification and transplantation was actually found to be the most deleterious combination with respect to the follicle reserve, and even worse when culture with Akt stimulators was performed.

State of the Art in Fertility Preservation for Female Patients Prior to Oncologic Therapies

“…SonerCelik et al [60] research team's findings regarding ovarian cryo-preservation and auto-transplantation demonstrate that expression of inhibitor proteins that control primordial follicle reserve decreases in cryopreserved ovaries after transplantation. The observation is consistent with the ovarian activity rush observed by others [61,62], and thus they debate the recommendation of follicular activation prior (in vitro activation IVA) to transplantation [58,63]. The longevity of the transplanted ovarian tissue varies widely and may depend on the age of the woman at cryo-preservation [64], some blaming the revascularization rate after transplantation as an important issue [65]; however, long functioning viability of more than ten years has been reported [66].…”

In vitro activation of ovarian cortex and autologous transplantation: A novel approach to primary ovarian insufficiency and diminished ovarian reserve

“…Moreover, its applicability has been criticized in different aspects. First, it has been associated with a deleterious effect on the ovarian reserve because of a massive and premature follicular activation compromising the development of growing follicles and leading to atresia (‘burn out effect’) ( Grosbois and Demeestere, 2018 ; Dolmans et al , 2019 ). Second, the pathway that becomes activated from this technique has been found altered in several cancers ( Luongo et al , 2019 ), and also showed an increase in DNA damage with a difficult repair when it was applied in bovine follicles culture ( Maidarti et al , 2019 ).…”

“…Even so, histological samples reveal that the pool of follicles will not be completely exhausted until the entry into the eighth decade of life ( Griesinger and Fauser, 2020 ). The main reason for this event is atresia; however, most of them remain in a dormant state caused by certain inactivating factors, while those selected to grow will develop toward the primary stage under control of different signaling pathways ( Dolmans et al , 2019 ).…”

Pretreatment of ovaries with collagenase before vitrification keeps the ovarian reserve by maintaining cell-cell adhesion integrity in ovarian follicles

“…As a consequence, secondary follicles collected from frozen-thawed ovaries that were pretreated with collagenase showed normal follicular development. Vitrification of ovaries is known to promote an atretic process in most follicles, except for primordial follicles 19 . However, pretreatment with collagenase prevents atresia and enables the maintenance of developmental competence of secondary follicles.…”

“…However, after transplantation the function of frozen-thawed ovarian tissue preserved by vitrification did not improve compared to that of the slowly-frozen ovaries 18 . Furthermore, most follicles, except for primordial follicles, undergo atresia following transplantation or culture 19 .…”